Written By: Heather Brown-Harding, Ph.D.
When you need better resolution than what can be achieved using a traditional microscope, it can be very intimidating to figure out which microscopes will work best for your experiment. Super-resolution imaging methods require software reconstruction after image acquisition. This is because multiple images are acquired, and they need to be combined. Additionally, the points of light need to be reassigned to their true location.
Today, we’re going to discuss 5 different super resolution methods their pros and cons. Although Rayleigh Criterion is not broken, these techniques each feature creative ways to get around it.
1. Structured illumination microscopy (or SIM).
SIM uses polarized light sources, like lasers, filtered through diffraction grating. The orientation of the grating is changed several times, and the resulting multiple images are used to reconstruct an image with better resolution than any of the originals.
The points of lights, which would normally interfere with their neighbors, are excited and captured at different times. This allows 100-nanometer x-y resolution, and 300-nanometer z resolution. Due to the gentle nature of the technique and speed, SIM represents an ideal method for the imaging of live cells and small organisms. It is often used for long-term imaging of embryo development experiments using Drosophila or C. elegans.
SIM’s major disadvantage is that the resolution is only approximately twice that of confocal microscopy. However, it does produce good resolution, and rapidly images very large areas.
2. Stochastic optical reconstruction microscopy (STORM)
Nikon’s MicroscopyU describes STORM’s fundamental principle as follows:
“The activated state of a photoswitchable molecule must lead to the consecutive emission of sufficient photons to enable precise localization before it enters a dark state or becomes deactivated by photobleaching.”
This simply means that you will be imaging fluorophores that randomly light up, allowing for the reconstruction of a single image from a large number of images with sparse labeling. Since the sample is sparsely labeled, it is unlikely that two nearby molecules will admit light at the exact same time. In this way, the software can more accurately determine both the location of a molecule and reconstruct an image with a 10-to-55-nanometer resolution in the x-y and z.
The acquisition takes several minutes per image, and only some dyes are able to blink. Recent advances have made it possible to utilize STORM with live cell imaging, but it’s not ideal for highly dynamic processes or experiments that run for long durations.
3. Photo-activated localization microscopy (PALM).
Eric Betzig is credited with the development of PALM, and he won the Nobel Prize for his work. PALM is in the same family as STORM, as they both use blinking fluorophores as well as multiple images to create a single image. The difference between the two is that STORM uses dyes, while palm uses photo-activatable fluorescent proteins. The high-end density of light needed for many frames means that PALM is not ideal for dynamic processes. There is a great YouTube video of the Eiffel Tower at night.
4. Stimulated emission depletion (STED).
Stefan Hell won a Nobel prize for developing this technique. The distilled version of STED is that you illuminate a spot approximately 200 nanometers wide with a green light, just like that of a laser scanning confocal. The difference is that a donut-shaped red light is projected onto the same spot, turning off all the fluorophores except for a very small area in the center of the hole of the donut-shaped beam. Because the microscope knows exactly where a point of light came from, the researcher can achieve superior resolution.
Some experts with ideal samples can achieve a resolution of just a few nanometers, but it’s more realistic to expect a 30-nanometer resolution. Since this method is just an extension of the laser scanning confocal, it pairs well with live cell imaging. Generally, though, you will get slightly less resolution than you would with STORM or PALM.
5. 4Pi microscopy
4Pi microscopy is used for the increase of an image’s axial resolution. Normally, the axial resolution (or z resolution) is about 500 nanometers due to the structure of the point spread function.
With 4Pi microscopy, two objectives are utilized on either side of the sample, and only the illuminated part of the sample, which is in focus for both objectives, is kept. Thus, the resolution in the z-direction is about 100-150 nanometers. This technique is very useful for thicker samples like spheroids that can get severely distorted in the z-direction using conventional confocal microscopy. 4Pi doesn’t have the same super resolutions, but it’s definitely improved 3D imaging.
An instructive paper by Sage et al. goes over the different imaging and software performed on real data. Check it out to get the flavor of some of the different benefits of each technique.
To learn more about how to Use These 5 Techniques for Super Resolution, and to get access to all of our advanced microscopy materials including training videos, presentations, workbooks, and private group membership, get on the Expert Microscopy wait list.
In her free time, she enjoys playing with her cat & dog, trying out new craft ciders and painting.You can find her on twitter (@microscopyEd) a few times of day discussing new imaging techniques with peers.
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