User:Srustavi/Super resolution microscopy

Introduction

Due to the diffraction of light, the resolution of conventional light microscopy is limited as stated by Ernst Abbe in 1873. A good approximation of the resolution attainable is the FWHM (Full Width at Half Maximum) of the Point Spread Function. Techniques that break the resolution limit have been realized and they include STED , GSD, SSIM, PALM, FPALM, STORM and many others.

STED

STED (Stimulated Emission Depletion Microscope) uses two laser pulses, the excitation pulse for excitation of the fluorophores to their fluorescent state and the STED pulse for the de-excitation of fluorophores by means of stimulated emission.In practice, the excitation laser pulse is first applied whereby a STED pulse soon follows. Furthermore, the STED pulse is modified in such a way that it features a zero-intensity spot, which coincide with the excitation focal spot. Due to the non-linear dependence of the stimulated emission rate on the intensity of the STED beam, all the fluorophores around the focal excitation spot will be in their off state (the ground state of the fluorophores). By scanning this focal spot one retrives the image. The FWHM of the PSF of the excitation focal spot can theoretically be compressed to an arbitrary width by raising the intensity of the STED pulse, according to equation (1).

$\Delta r\approx {\frac {\Delta }{\sqrt {1+I_{max}/I_{s}}}}$   (1)

∆r is the lateral resolution, ∆ is the FWHM of the diffraction limited PSF, Imax is the peak intensity of the STED laser and Is is the treshold intensity needed in order to achieve saturated emission depletion.

The main disadvantage of STED which has prevented its widespread use is that the machinery is complicated. Also, the image acquisition speed is slow because of the need to scan the sample in order to retrieve an image. Due to a large Is value associated with STED, there is a need for a high intensity excitation pulse which may cause damage to the sample.

GSD

GSD microscopy or Ground State Depletion microscopy, uses the triplet state of a fluorophore as the off - state and the singlet state as the on – state, whereby an excitation laser is used to drive the fluorophores at the periphery of the singlet state molecule to the triplet state. This is much like STED ,where the off-state is the ground state of fluorophores, which is why equation (1) also applies in this case. The Is value is smaller than in STED making super resolution imaging possible at a much smaller laser intensity. Compared to STED though, the fluorophores used in GSD are generally less photostable and the saturation of the triplet state may be harder to realize.

SSIM

SSIM (Spatially Structured Illumination Microscopy) exploits the nonlinear dependence of the emission rate of fluorophores on the intensity of the excitation laser. By applying a sinusoidal illumination pattern with a peak intensity close to that needed in order to saturate the fluorophores in their fluorescent state one retrieves moiré fringes. The fringes contain high order spatial information which may be extracted by computational techniques. Once the information is extracted a super resolution image is retrieved.

SSIM requires shifting the illumination pattern multiple times, effectively limiting the temporal resolution of the technique. In addition there is a need of very photostable fluorophores due to the saturating conditions. These conditions also induce radiation damage to the sample which restricts the possible applications in which SSIM may be used.

PALM,FPALM and STORM

Given an isolated fluorescent molecule, one is able to determine its location with arbitrary precision according to equation (2).

$\Delta loc\approx {\frac {\Delta }{\sqrt {N}}}$   (2)

Δloc is the localization precision, Δ is the FWHM of the PSF and N is the number of collected photons.When dealing with a fluorophore-labeled specimen though, this cannot be done due to the dense packing of fluorophores and consequently overlapping images. But what if the fluorophores can be sequentially activated? Then the location of each activated fluorophore may be determined and a super-resolution image may be obtained. This is how PALM(Photo-Activated Localization Microscopy) ,FPALM (Fluorescence-PALM) and STORM (Stochastical Optical Reconstruction Microscopy) works.

In these methods, some fluorophores are first activated stochastically with an excitation laser such that, on average, the distance between the activated fluorophores are larger than the FWHM of diffraction-limited light. Then the activated fluorophores are continually excited whereby the emitted photons are collected and the location of the fluorophores are determined with a precision given by equation (2). This process is repeated and a super-resolution image is constructed from the location of all individual fluorophores.

The desirable traits of the fluorophores, in order to maximize the resolution, are that they should by bright. That is, they should have a high extinction coefficient and a high quantum yield. They should also possess a high contrast ratio (ratio between the number of photons emitted in the light state and the number of photons emitted in the dark state). Also, a densely labeled sample is desirable according to the Nyquist criteria.