Abberior Instruments

Introduction

Separate labeled features that are closer than the diffraction barrier

Individual dye label
Diffraction area of visible light
Individual dye label
Label in fluorescent mode
Diffraction limited area of excitation

Due to diffraction there is always an area larger than half of the light wavelength illuminated; all molecules in this area emit light and cannot be distinguished.

Keep adjacent fluorophors sequentially dark, so that one can register them separately und thus record adjacent features sequentially.  Thus, the basic idea of nanoscale resolution is to introduce a controllable on/off transition of the dye label.

Consequently, the dye is the key for nanoscale resolutionAbberior is specialized in designing dyes with on/off transitions that can be controlled via a second light source.

A: Coordinate-Targeted Readout

  • The sample is scanned in a predetermined pattern with the excitation/deexcitation pattern being moved across the sample.
  • Each point in the sample must be addressed at least once, but several scans may be collected to improve the signal.
  • Only molecules in the very center of the excitation area remain in the on state.
  • Molecules need to be switched between on and off multiple times while readout.

STED: STED light forces the molecules into the ground state via stimulated emission. Thus fluorescence cannot occur under STED irradiation which prepares a controllable 'off'-state

RESOLFT:Generalized concept of switching all previously excited molecules off, except the very center of the focus. A particular implementation concept is a light controllable conformational change of the molecule. One isomerized form represents the non-fluorescent 'off'-state


B: Spatially Stochastic Readout

  • Each molecule must be photoactivated and deactivated at least once; multiple switching cycles are possible but not mandatory.
  • The readout order is random (molecules are randomly switched on) and does not follow any predetermined sequence.
  • The molecule positions are calculated from individual image frames and are used to synthesize the final high-resolution image.

PALM, STORM: Molecules are randomly switched into the 'on'-state, while most molecules stay in the 'off'-state at a certain point of time. Switching different molecule subsets is realized either reversably (e.g. conformational changes) or irreversably (e.g. uncaging)

GSDIM: In contrast to the previous implememtation GSDIM (also referred to as dSTORM) realizes switching of molecule subsets via exploiting purely electronic states of a fluorescent molecule. An electronical dark-state with a sufficiently long lifetime represents the 'off'-state