Gated-STED

2colourSTEDStimulated emission depletion (STED) microscopy is a super-resolution technique capable of below 50nm resolution through the selective depletion of fluorescent molecules using two synchronised lasers. Improved resolution can be acheived in 3 Dimensions, in up to 4 colours and in live samples.

Gated-STED is performed on a Lecia SP8 STED 3X microscope (see specification) equipped with a super continuum white light laser, a CW-592nm and pulsed-775 nm depletion laser and a 405 nm diode laser. This system is also equiped with Hyvolution for improved resolution in confocal imaging.

The Leica Sample Guide can help you choose the best dyes: for the ESRIC SP8 3X STED any that are depleted by 592 nm and/or the 775 nm line are appropriate (not the 660 nm line).

This microscope is available at Heriot-Watt University site, for all enquiries please contact Ali Dun This email address is being protected from spambots. You need JavaScript enabled to view it.. Image credit: Andrew Jarjour, SCRM, University of Edinburgh

SIM                                                                                

SIM scaledNikon N-SIM Structured Illumination Microscopy (SIM) utilises a structured light pattern to image fluorophores within a biological specimen in 3D at double the conventional resolution. This technique can achieve up to 120 nm in xy and 300 nm in z (sample dependent) in 4 colours.

This super resolution technique is performed on a Nikon N-SIM system (see specification).  

This N-SIM system microscope is available at the University of Edinburgh site, contact Ann Wheeler This email address is being protected from spambots. You need JavaScript enabled to view it..

Image credit Alan Egan, ESRIC PhD Student

SRRF-Stream

Andor SRRF-Stream is a commercial implementation of the SRRF algorithm (Super Resolution Radial Fluctuations) developed by Ricardo Henriques at University College London.  By capturing many images of the same field it is possible to calculate a probabilistic estimate of the position of fluorophores by analyzing the radial intensity gradient and the intensity fluctuations characteristic of these molecules.

The advantage of SRRF is that it is one of the few super resolution methods conducive to live cell imaging.  Conventional fluorophores can be used with comparatively low excitation energies from widefield or laser based sources with image capture rates of 1-2 seconds per frame.

SRRF is best suited to samples where the fluorophore density is low or higher density samples where photoswitchable fluorophores are used in the presence of a reducing buffer environment.  Depending on the sample, the equivalent lateral resolution afforded by SRRF can be 50-150nm.

SRRF-Stream is available on the Andor Dragonfly Spinning Disk confocal microscope utilizing widefield, TIRFM or confocal modalities (see specification).

The microscope is available at the University of Edinburgh (IGMM) site.  Please contact Ann Wheeler This email address is being protected from spambots. You need JavaScript enabled to view it. for more information.

A live capture of mitochondrial movement in a COS 7 cell.