You might have heard about the new techniques that fall under the umbrella of ‘Super-Resolution Microscopy’. In this article, we’ll look at diffraction limits and how to overcome them using Super-Resolution Microscopy techniques.
The Limits of Light Microscopy
Light microscopy is one of the most versatile tools in a biologist’s arsenal, but from its invention in the 17th Century until a few decades ago, it was constrained by a seemingly impenetrable barrier: the diffraction limit. Simply put, the diameter of the smallest discernible object is equal to the wavelength used to observe it divided by twice the numerical aperture (NA) of the microscope.
With visible light, the smallest usable wavelength is around 450 nm (blue), and the largest NA possible with current technology is around 1.4. Even with advances such as confocal microscopy and deconvolution algorithms, only biological structures larger than 200 nm could be visualized.
Sub-diffraction limit techniques were postulated in the early 20thCentury, and while significant theoretical advances were made starting in the late ‘70s, only in the last decade have these technologies become available to the average researcher.
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Breaking the diffraction barrier- a menagerie of options
There are three major families of super-resolution light microscopy;
- localization techniques
- structured illumination microscopy (SIM)
- stimulated emission depletion (STED)
In addition to covering these techniques in this article, we’ll also look at a handful of other technologies known as “Near-field Methods”.
1. Single Molecule Localization Techniques
STORM, dSTORM, PALM, fPALM, PALMIRA, SPDM, GSDIM, BaLM, and PAINT are all included in this category. Using a conventional or TIRF light path, various strategies are used to collect only a small fraction of the fluorophores marking your structure of interest in any one image. This is repeated hundreds of times, and each fluorophore is assumed to represent the smallest possible object, as delineated by its point spread function (PSF) in post-processing. From these thousands of individual points, the full image can be assembled.
- Resolution: Lateral resolution (in the XY plane) approaching 20-50 nm. Axial (in the Z plane) resolution is limited to ~200 nm away from the coverslip, and can be improved to 50-80 nm. Low labeling density is critical for the best resolution.
- Pros: Theoretically single-molecule precision, excellent delineation of small, punctate, or filamentous objects such as discrete macromolecular complexes.
- Cons: Long imaging times (often in the tens of minutes), frequent requirement of fluorophores with special optical properties, and limitation to essentially 2-D imaging near the coverslip.
- Applications: Membranes; receptors; adhesion complexes; protein clustering and movement. See Sengupta et al., 2012 for further details.
2. Structured Illumination Microscopy (SIM)
SIM works by creating patterns in the amplitude and phase of widefield excitation light before it reaches the sample. Interference between those patterns and patterns generated by the sample are used to algorithmically reconstruct the sample’s sub-diffraction structure. By including information from above and below the focal plane (3-D-SIM), axial resolution is improved and can image the entire axial dimension of cultured cells.
- Resolution: ~100 nm laterally and ~200 nm axially.
- Pros: Can use conventional fluorophores, has fast multicolor imaging and improved axial resolution. ~10 micron sampling depth is well suited for imaging 3-D structures.
- Cons: Particularly susceptible to artifacts caused by sample structure, preparation, and system errors. Has the lowest lateral resolution of all super resolution techniques.
- Applications: Chromatin structure; chromatin modifiers; nuclear pores; actin and tubulin networks; live-cell applications; compatible with other techniques such as FISH (Markaki et al., 2012).
3. Stimulated Emission Depletion (STED)
STED relies on using a second laser beam in a scanning confocal system to deplete emission fluorescence around the point being illuminated. Imagine that with a normal confocal scanning laser microscope you can image a point of a certain diameter. Now imagine a doughnut-shaped second laser beam of a different wavelength suppressing the excited fluorophores, leaving only the ‘hole’ in the doughnut to be collected by the detector.
- Resolution: ~50-80 nm lateral resolution, ~500-800 nm axially – the same as a standard confocal microscope.
- Pros: High lateral resolution with a relatively conventional setup, excellent imaging depth (>20 microns), few artifacts since no algorithms are required.
- Cons: Limited fluorophore options, relatively slow speeds (as with confocal), a maximum of two colors with no 405 nm option, the high energy required can cause bleaching and sample degradation.
- Applications: Vesicles; membranes; synaptic behavior. See Müller et al., 2012 for further details.
4. Near-field Methods
Total internal reflection (TIRF) microscopy has been available for some time and utilizes an optical phenomenon at the interface of materials with divergent refractive indices. Because this phenomenon (an evanescent wave) only occurs in the first 100-200 nm beyond the coverslip, you are effectively achieving axial super resolution, but are limited to structures that exist at the coverslip.
A more advanced method, near-field scanning optical microscopy (NSOM, SNOM) uses the TIRF principles but forgoes an objective lens for a very fine physical aperture such as the end of a glass fiber passed over the sample, and can achieve lateral resolution up to 20 nm. However, this is still limited to structures on the surface of the cell and can be difficult to implement.
5. Dual-Objective Methods
Most of the methods above can be combined with an interferometric approach of two opposing objectives to improve axial resolution. The first approach is 4Pi microscopy, which aligns two objectives that can together act as a single lens and reach axial resolution of ~80 nm.
The same principle is applied to several other modalities: widefield imaging in I5M or SMI; structured illumination in I5S; STED in isoSTED; and PALM in iPALM. All of these techniques are quite technically challenging, as they are dependent on exceptionally precise alignment and are quite sensitive to sample aberration and temperature changes.
It’s both better and worse than it seems
Two things should always be kept in mind when considering your options in super-resolution microscopy. Firstly, the technology is constantly advancing and some the statements in this introduction may have become obsolete by the time you read it- for example, localization techniques are experiencing rapid improvements at multi-channel acquisition and Z resolution.
Secondly, many of the resolutions (and a few of the techniques cited) are ceilings which are achieved in highly specialized labs with custom-built systems that may not be practical for the average researcher. Most of the techniques discussed here are available commercially, but performance can vary.
You may be tempted to go and lobby for your very own super-resolution system immediately, but remember that there are many things to take into account. Resolution is only part of the equation- you may value multi-color imaging, in vivo observations, high-speed imaging, three-dimensional imaging, or low photon intensities. Each priority comes with a compromise in another area, and the optimal technique will be entirely dependent on the specific demands of your experiment.
Get started!
Once you’ve digested all of that, you can proceed with acquiring fantastic images. Your best bet is to visit your institution’s imaging core and talk to the manager. They are best equipped to help you decide which technology is most suited for your experiment, what reagents you may need, how to prepare the samples, and how to navigate image acquisition and post-processing. It may seem like a lot of work, but once you’re generating data and seeing structures at sub-200 nm resolution, we guarantee you’ll be hooked!
For a closer look at one fast variant of Super Resolution Microscopy, see multifocal structured illumination microscopy.
References
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