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STED microscope

Stimulated Emission Depletion microscopy

A Stimulated Emission Depletion (STED) microscope has been designed and built by the Laser Analytics Group. The system's development has been funded by the MRC's Next Generation Optical Microscopy Initiative. The microscope is currently in development and being tested on a number of biological samples. For further information on the status of the microscope and the possibility of  please contact Dr Pierre Mahou.

Figure 1
Figure 1. Principle of Stimulated Emission Depletion microscopy (STED).

Principle

STED microscopy shares principles of confocal laser scanning microscopy and structured illumination microscopy.  In traditional confocal microscopy a laser beam is focused to a diffraction limited spot, referred to as the point spread function, PSF.  The PSF has a diameter of ca 250 nm and excites fluorescence in the sample, which is collected as the beam is raster-scanned across the region of interest (Figure 1a).  One can think of the laser beam as a paint brush - the thinner the tip, the finer the detail we can recover from the sample ('Excitation beam' shown in blue in figure below). The trick of STED, or more generally the RESOLFT principle, is to 'shape' the tip into a spot much smaller than the 250 nm diffraction limit, by using a so-called depletion beam, which has a doughnut shaped intensity distribution, with zero intensity in the middle and high intensity in an annular region around the central position ('STED beam' in the figure below).  Superimposing this depletion beam on the excitation beam prevents spontaneous emission (fluorescence) from fluorophores anywhere except in a very small central region, thus yielding an effective PSF of only 30 to 90 nm in diameter (Figure 1b-c).  This permits one to gain information on a much smaller scale than possible with standard confocal imaging. The technique works at a reasonable depth and is thus useful to look at distribution of molecules deep within cells.

Figure2Bis.jpg
Evaluation of the performance of the lab-built STED microscope from ideal calibration samples. (a) Estimation of the resolution of the confocal/STED modality from individual antibody cluster (dye STAR 635P). The resolution enhancement is on the order of 3. (b) Performance of the STED modality in a case of a dense sample formed by 100nm beads. (c) Confocal/STED images of amyloid fibrils labelled with the dye STAR 635P.

Application of STED microscopy to study amyloid fibrils formation

The developed super resolution optical microscope based on STED has been used to study amyloid fibrils labelled with red dyes such as the ATTO or Aberrior dyes. The excitation of various fluorophores is enabled by a supercontinuum source generated by pumping a photonic crystal fiber and the STED beams is produced by a spatial light modulator that also corrects for the optical aberrations of the system. This microscope has been successfully evaluated with ideal test samples (Figure2a-b) and is now being used to monitor the formation of amyloid fibrils in vitro (Figure 2c).