Microscopes

DeltaVision OMX inverted

The DeltaVision OMX is primarily a super-resolution microscope system. Recent advances in microscopy have permited the diffraction barrier to be bent or broken(1). Most optical microscopes are limited in resolution to~250 nm, however the new techniques can surpass that many times over. The OMX has two such techniques:

  1. 3D Structured Illumination Microscopy (3D-SIM) and
  2. a single molecule localization technique called DLM.

Due to the optical and computational design of the OMX it is also able to acquire incredibly quick multi-channel 3D widefield or 2D TIRF timelapse movies. The system is equipped with a stage heater, objective heater and chamber to deliver humidified CO2-rich air.
 
3D Structured Illumination Microscopy
3D-SIM can give 110-130 nm lateral resolution and 280-350 nm axial resolution in 3 colours using common fluorophores. Together this offers an 8-fold increase in volumetric resolution. One advantage of 3D-SIM over other super-resolution techniques is that normal sample preparation techniques can be used. Please refer to this document for our recommendations on sample preparation.
 
During 3D-SIM acquisition a structured illumination pattern is rotated and moved in phase through multiple acquisitions (as shown in the movie below). 15 images are acquired per slice - 3 angles and 5 phase shifts. The interaction of the structured illumination pattern with the structures in the sample produce moiré fringes which encode the higher frequency information present within the sample.

3D-SIM acquistion

A reconstruction algorithm is then applied to produce images at a higher resolution. The microscope and technique was developed by John Sedat, David Agard and Mats Gustafsson(2,3) and has been commercialised by Applied Precision (now part of GE Healthcare).

The difference between a widefield fluorescence and a 3D-SIM image

DLM Localisation Microscopy
Monet acquisition relies on collecting many thousands of images of blinking of fluorescent molecules. The blinking comes through the manipulation of the illumination regime and/or sample preparation. The image sequence is then put through a computational localization step to acheive 20-50 nm lateral resolution. In practice this is done with TIRF illumination so that only fluorophores at the coverslip surface are excited.

DLM acquisition

A localised Monet image

Our OMX Specification:

 

Installation of the IMB OMX over 4 days

images acquired every 30 seconds

Further reading

More on GE Healthcare's cellular imaging and analysis product range:

 

References
(1) Schermelleh, et al. (2010) A guide to super-resolution fluorescence microscopy. J. Cell Biol. 190:165-175 PUBMED
(2) Gustafsson (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc. 198:82-87 PUBMED
(3) Gustafsson, et al. (2008) Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophy. J. 94:4957-4970 PUBMED