Super-Resolution Technology
What is super-resolution microscopy?
In recent years a variety of new approaches such as Structured Illumination (3D-SIM), Localization Microscopy (PALM, STORM) and Stimulated Emission Depletion (STED) have been developed to surpass the limits of conventional optical microscopes. Collectively referred to as super-resolution microscopy, these methods allow precise visualization and measurement of features that are below the diffraction limit.
Structured Illumination Microscopy (3D-SIM)
Three Dimensional Structured Illumination Microscopy (3D-SIM) projects a structured light pattern onto the sample. The illumination pattern interacts with the fluorescent probes in the sample to generate interference patterns know as moiré fringes. By modulating the illumination pattern, collecting and reconstructing the subsequent images, super-resolution images with double the lateral and axial resolution are obtained.
3D-SIM techniques work with traditional fluorescent proteins and dyes commonly used in fluorescent imaging. In addition, 3D-SIM imaging is not limited to regions of interest at the coverslip and can image up to 10 microns past the coverslip into the sample.
Figure 1. 3D-SIM Pattern generation in DeltaVision OMX imaging system.
Localization Microscopy (LM)
Localization microscopy identifies the position of individual fluorophores by imaging a few at a time using photoactivable or photoswitchable fluorophores. When only a single fluorophore is emitting light, the precise position of that fluorophore can be determined by fitting a Gaussian curve to the spot and assigning the peak of that curve to represent the location. Challenges arise when multiple fluorophores are too closely positioned such that images overlap and single molecules can no longer be resolved.
Commercially available Localization Microscopy methods are implemented in Total Internal Reflection Fluorescence (TIRF) Microscopy mode and rely on the use of either specialized dye pairs (STORM) or photo-switchable fluorescent proteins (PALM). While these methods can increase the lateral resolution by an order of magnitude over wide-field methods, they are largely limited to one or a few optical sections due to limitations in the TIRF method. Furthermore, they require that a very limited number of fluorescent molecules be activated in each cycle of imaging to minimize the number of overlapping fluorophores in each image. This places limits on labeling density and image acquisition speed with most configurations. This issue has been addressed in Applied Precision’s new Monet™ imaging method.
Stimulated Emission Depletion (STED) microscopy was developed by Stefan Hell and colleagues at the Max Planck Institute in Göttingen, Germany. STED effectively manipulates the area generating fluorescence by shaping the excitation spot using emission depletion. The resolution of STED imaging can be varied by adjusting the power of the depletion laser and in biological samples is tuned for lateral resolution on the order of 80nm. It should be noted that the shaping of the excitation spot only achieves resolution improvement in the lateral direction.