The diffraction-limited nature of light microscopy does not always provide sufficient resolution to see all desired details. At the AOMC, multiple techniques are available to record images with improved (super) resolution. Each of these methods has its own set of advantages and disadvantages, so they can be matched to almost any research question.
SIM is capable of providing a doubled spatial resolution by using a patterned (structured) illumination, usually stripes, to excite the sample. The interaction between the illumination pattern and the sample-structure moves high frequency information, which would normally not be resolvable, into a resolvable range. In practice, a series of 15 - 25 images is acquired while rotating and shifting the illumination pattern and the super-resolved image is subsequently calculated.
The low number of input images makes SIM a fast super-resolution microscopy method. Additionally, no special fluorophore (no "blinking") is required, which means it can be used for many samples and fluorophore combinations. The method does, however, only offer a twofold resolution improvement and can be prone to artifacts when out-of-focus light or refraction index mismatch is present.
|fast (< 1s)||"only" a twofold resolution improvement|
|no special fluorophores required||limited penetration depth (widefield system)|
|suitable for live samples||(prone to artefacts)|
SMLM groups a collection of microscopy techniques that rely on separating and fitting the emission of individual emitters (single molecules) to determine their exact location. Type examples include photo-activatable localization microscopy (PALM), direct stochastic optical reconstruction microscopy (dSTORM) and point accumulation for imaging in nanoscale topography (PAINT). The first two methods use sequential photo-activation or -conversion between two distinguishable states of the employed fluorophores to only have a small subset of fluorophores emitting in the detection channel at any single point in time. The last method uses dyes that are minimally fluorescent when freely diffusing, but brightly fluorescent when bound or immobilized by the structure of interest. In all cases, large timeseries of widefield fluorescence images of sparse emitters are acquired and post-processed to obtain very high resolution images.
SMLM is capable of achieving very good spatial resolution, close to 10-fold better than the resolution limit, but often sacrifices temporal resolution to achieve this. In many cases, the best results are obtained with fixed samples.
|very high resolution obtainable (tenfold improvement)||slow (> 5min)|
|requires specific fluorophores (and buffer conditions)|
|limited penetration depth (best results with TIRF)|
|mostly fixed samples|
SOFI is a post-processing method to extract super-resolution information in three dimensions from a timeseries of widefield fluorescence images. The method analyzes temporal fluctuations in the intensity of fluorophores (aka "blinking"). While SOFI provides a theoretically unlimited resolution improvement, in practice it is more commonly used to provide a two- to threefold resolution enhancement. This resolution improvement can be achieved with a relatively short timeseries (a few hundred to a few thousand frames) and a relatively poor signal-to-noise ratio. Additionally, because the method does not rely on the identification of single emitters like the SMLM methods described above, a higher fluorophore density can be used. The combination of these factors makes SOFI a robust method for the acquisition of medium-high resolution images (80 - 180 nm resolution).
|medium fast (s - min)||medium fast (s - min)|
|medium resolution gain (twofold - fourfold)||medium resolution gain (twofold - fourfold)|
|suitable for live samples||requires specific fluorophores|
|suitable for low SNR samples|
A commercial implementation of the Image Scanning Microscopy (ISM) technique to improve the spatial resolution of confocal laser scanning microscopy by up to a factor of two. The method employs a grid of detectors, each of which acting as a tiny confocal pinhole, to detect the entire emission spot. The detected fluorescence is reassigned in a post-processing step to a more narrow (higher resolution) spot resulting in the improved resolution image.
Airyscan imaging allows the use of a confocal microscope as if it had a very small pinhole, thus unlocking its true resolution potential, without compromise to the speed or sensitivity. Moreover, the microscope can be configured in such a way to target an improved resolution, an increased signal-to-noise ratio or a very fast imaging speed ("Fast Airyscan").
|very versatile (balance speed, resolution and SNR)||"only" up to a twofold resolution improvement|
|no drawbacks compared to confocal microscopy||medium speed (confocal speed)|
|compatible with two-photon excitation|
|suitable for live samples|