Super-resolution microscopy allows images to be taken with a higher resolution than an optical microscope’s theoretical limit.
The minimum separation between two light sources that can be resolved into different objects is governed by the Rayleigh criterion. This places a theoretical limit (“diffraction limit”) on the resolution of images. Super-resolution microscopy is used to resolve fluorophores separated by a distance smaller than the diffraction limit. The techniques used to achieve this include STED, PALM, STORM, and SIM.
Stimulated Emission Depletion (STED) microscopy
STED microscopy resolves features that are smaller than the diffraction limit using a pair of synchronised pulsed lasers (an excitation laser and a depletion laser).
The excitation pulses are reflected from a dichroic mirror towards an objective lens and onto a sample. Fluorophores illuminated by the laser spot absorb this light and fluoresce.
The depletion light is phase-modulated to generate ring-shaped pulses, and then reflected onto the same region of the sample using another dichroic mirror. The red-shifted wavelength of the depletion light stops fluorescence by forcing stimulated emission. This occurs in the outer region of the ring, allowing the centre to fluoresce. The depletion pulses are therefore shaped to minimise the effective size of the excitation beam, allowing features smaller than the diffraction limit to be resolved.
The objective lens used to illuminate the sample collects the fluorescent light which then passes through both dichroic mirrors and onto a detector.
Photo-Activated Localisation Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM)
PALM and STORM are techniques that capture and localise the fluorescent emissions of just a few isolated fluorophores in a sample.
The fluorophores are repeatedly excited (“photoactivated”) until the intensity of their emissions can be accurately modelled by a Gaussian function. The density of fluorophores is kept sufficiently low so that the overlapping of their emissions is minimised. The centre of each Gaussian function is then localised, and the location of the fluorophores can be mapped.
PALM and STORM achieve single molecule localisation using repeated photoactivation cycles. PALM typically uses fluorescent proteins that eventually photobleach (irreversible damage causing an inability to fluoresce) after many exposures. STORM is used to control the emission of synthetic fluorescent dyes that can reversibly switch between emissive and non-emissive (or less emissive) states.
Structured Illumination Microscopy (SIM) generates a super-resolved image by exciting a fluorescent sample using a series of patterned excitation lines and computationally superimposing the resulting fluorescence signals.
Structured Illumination Microscopy (SIM)
SIM uses patterned lines of excitation light that are rotated to generate a series of images with high spatial frequency.
The excitation light passes through a diffraction grating to create an interference pattern. The light is then focussed by an objective lens and onto the sample. Fluorophores in the sample absorb the light and fluoresce.
A series of interference patterns are created by rotating the grating in discrete steps. Each interference pattern is phase-shifted multiple times to excite more fluorophores.
The fluorescence emission in each configuration is imaged by a camera. The images are then superimposed to create a super-resolved image.